In principle, differentiated cardiomyocytes derived from hESCs and hiPSCs are ideal model systems in which to study the physiological effects of disease and aging. However, hESC/hiPSC-derived cardiomyocytes characteristically exhibit electrical and mechanical properties of early fetal, rather than adult, cells. Although a few growt factor-like molecules are reported to have a maturational effect, little is known about the precise physiological effects or how the molecules can be used to improve the utility of hiPSC/hESC-derived cardiomyocytes to model aging and disease. The goal of this R21 proposal is to identify and characterize such molecules, based on preliminary data that endothelial cells normally produce maturation-inducing factors. Data are presented showing the development of instrumentation and software for high-throughput assessment of electrophysiological maturation. Using this technology, we provide preliminary evidence that endothelial cells (ECs) promote electrical and ion channel profile maturation of hiPSC/ESC-derived cardiomyocytes through the production of factors that accumulate in conditioned media. The specific aims of this proposal are to: 1) define EC-induced maturation by functional expression of ion channels and pumps, drug sensitivity profiling, action potential and calcium handling metrics, 2) determine if ECs also promote maturation of contractile apparatus structure and function, and 3) use the electrical and mechanical metrics from Aims 1 and 2 as quantitative metrics to identify and characterize the effects of EC-derived maturation factor(s). The identification of signals that promote cardiomyocyte maturation should lead to improved methods for deriving functional cardiomyocytes that will be enabling for studies of aging and disease. Beyond the high- risk/high-yield scope of the R21 project, we have established collaborations to evaluate whether the maturational factors indeed enhance the utility of hiPSC-cardiomyocytes as models for drug risk assessment and disease.